Pharmaceutical compositions containing fibroblast growth factor-related peptides

ABSTRACT

The present invention relates to a pharmaceutical composition comprising, as an active ingredient, a peptide represented by the following formula (I): 
     
       
         MTS-X-NLS  (I) 
       
     
     wherein MTS represents a membrane-transfer amino acid sequence; X represents a direct bond or a linker sequence; and NLS represents a nuclear localization amino acid sequence, or a pharmaceutically acceptable salt thereof, particularly, a pharmaceutical composition having food and water intake inhibitory effects, and accompanying weight-reducing and obesity-inhibiting effects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §371 tothe PCT International Application PCT/JP99/01697, filed Mar. 31, 1999,published Dec. 23, 1999 as WO 99/65512, in Japanese; which claimspriority to Japanese Application No. 172325/1998, filed Jun. 19, 1998.All applications are incorporated by reference in their entirety and forall purposes.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions comprisinga peptide derived from fibroblast growth factor as an active ingredient.

BACKGROUND OF THE INVENTION

Some Fibroblast Growth Factors (called FGF hereinafter) are known tohave various physiological activities, and a variety of studies havebeen made with the expectation of their use as pharmaceuticals. A foodintake inhibitory effect is known to be one of the physiologicalactivities of FGF. The FGF that is released from ependymocytes of thecerebral ventricle wall by responding to a glucose concentration in thebrain, is considered to act on neurons of the feeding center in thebrain repressively, resulting in the food intake inhibitory andweight-reducing effects.

Since FGF is a protein comprising one hundred and several tens of aminoacids, however, there were the following problems with its use, as is,as a pharmaceutical: 1) the synthesis of FGF is difficult; 2)immunological rejection occurs in vivo; 3) administration methods arelimited; 4) it is difficult to conserve FGF without causing changes ofthe active three-dimensional structure.

Therefore, there are strong demands on development of medicamentscomprising FGF with physiological activity, particularly, food intakeinhibitory effect, without any of the above problems.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a pharmaceuticalcomposition having physiological activity, particularly, food intakeinhibitory effect of FGF and having no problems that occurs when FGF isused directly as a medicament.

The inventors have studied extensively and intensively to solve aboveproblems and have now found that a partial peptide derivative,Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Val-Ala-Ala-Ala-Asn-Try-Lys-Lys-Pro-Lys-Leu(SEQ ID NO:3) (Lin Y. Z. et al., J. Biol. Chem., 270, 14255-14258(1995), of an FGF previously known to have superior cell membranetransfer and cell nuclear membrane transfer, unexpectedly has strongfood and water intake inhibitory effects. The inventors have furtherfound based on this finding that the peptide which is obtained bycombining a peptide with cell membrane transfer and a peptide with cellnuclear localization has strong food and water intake inhibitoryeffects, and accompanying weight-reducing and obesity-inhibitingeffects, thereby completing the present invention.

Thus, the present invention comprises the following inventions:

(1) A pharmaceutical composition comprising a peptide represented by thefollowing formula (I):

MTS-X-NLS  (I)

 wherein MTS represents a membrane-transfer amino acid sequence; Xrepresents a direct bond or a linker sequence; and NLS represents anuclear localization amino acid sequence, or a pharmaceuticallyacceptable salt thereof as an active ingredient.

(2) The pharmaceutical composition of (1) above, wherein the MTS isrepresented by the following amino acid sequence:

(a) an amino acid sequence of SEQ ID NO:1; or

(b) an amino acid sequence comprising deletions, substitutions oradditions of one or more amino acid residues relative to the amino acidsequence of SEQ ID NO: 1.

(3) The pharmaceutical composition of (1) or (2) above, wherein the NLSis represented by the following amino acid sequence:

(a) an amino acid sequence of SEQ ID NO:2; or

(b) an amino acid sequence comprising deletions, substitutions oradditions of one or more amino acid residues relative to the amino acidsequence of SEQ ID NO:2.

(4) The pharmaceutical composition of any one of (1) to (3) above,wherein the pharmaceutical composition is for inhibition of food intake.

(5) The pharmaceutical composition of any one of (1) to (3) above,wherein the pharmaceutical composition is for inhibition of waterintake.

(6) The pharmaceutical composition of (4) above, having aweight-reducing and/or obesity-inhibiting effect.

(7) The pharmaceutical composition of (5) above, having aweight-reducing and/or obesity-inhibiting effect.

The present invention will be described in detail as follows.

A pharmaceutical composition of the present invention is characterizedin that it comprises, as the active ingredient, a peptide represented bythe following formula (I):

MTS-X-NLS  (I)

where MTS represents a membrane-transfer amino acid sequence; Xrepresents a direct bond or a linker sequence; and NLS represents anuclear localization amino acid sequence, or a pharmaceuticallyacceptable salt thereof.

As used herein, the term “membrane-transfer amino acid sequence”represented by MTS means an amino acid sequence corresponding to apeptide with cell membrane transfer properties, and the term “nuclearlocalization amino acid sequence” represented by NLS means an amino acidsequence corresponding to a peptide with nuclear localizationproperties.

MTS may be any amino acid sequence as long as it is a membrane-transferamino acid sequence, but particularly preferred is the amino acidsequence of SEQ ID NO: 1 or an amino acid sequence comprising deletions,substitutions or additions of one or more amino acid residues relativeto the amino acid sequence of SEQ ID NO: 1.

As used herein, the term “one or more amino acid residues” does notlimit the number thereof so far as the cell membrane transfer of apeptide corresponding to the amino acid sequence is not lost.

The number of amino acid residues is, but not limited to, normally 7-40,preferably 16.

The NLS may be any amino acid sequence as long as it is a nuclearlocalization amino acid sequence, but particularly preferred is theamino acid sequence according to SEQ ID NO:2 or an amino acid sequencecomprising deletions, substitutions or additions of one or more aminoacid residues relative to the amino acid sequence of SEQ ID NO:2.

As used herein, the term “one or more amino acid residues” does notlimit the number so long as the nuclear localization of a peptidecorresponding to the amino acid sequence is not lost.

The number of amino acid residues is, but not limited to, normally 3-15,preferably 4-7.

In the formula (I), X represents a direct bond or a linker sequence. MTSand NLS may be bound together directly, or otherwise, a linker sequencemay intervene between MTS and NLS. The linker sequence includes, forexample, amino acid sequences comprising one or more Ala or Glyresidues, or combination thereof. The number of amino acid residues inthe linker sequence is, but not limited to, normally 1-20, preferably2-7.

The peptide represented by the formula (I) includes for example peptidesin which MTS is located on the N-terminal side while NLS on theC-terminal side, and peptides in which MTS is located on C-terminal sidewhile NLS on the N-terminal side.

The number of the amino acid residues represented by the formula (I) is,but not limited to, normally 10-40, preferably 23-27.

The peptides represented by the formula (I) can be chemicallysynthesized, for example, by known peptide synthesis methods.

Examples of peptide synthesis methods include azide, acid chloride, acidanhydride, mixed acid anhydride, DCC, activated ester, carboimidazole,oxidation-reduction, and DCC-additive (HONB,HOBt,HOSu) methods.(Schreder and Luhke “The Peptide” Vol.1 (1996), Academic Press, N.Y.,USA; Izumiya et al., “Peptide Synthesis”, Maruzen Inc. (1975)). Thesepeptide synthesis methods can be carried out either by a solid phasesynthesis or a liquid phase synthesis.

Examples of pharmaceutically acceptable salts of peptide which isrepresented by the formula (I) include sodium or hydrochloride salts.

As the pharmaceutical composition of the present invention, peptidesrepresented by the formula (1) or pharmaceutically acceptable saltsthereof may directly be used as they are, or alternatively they may beformulated in combination with pharmaceutically acceptable carriers.

Examples of pharmaceutically acceptable carriers include excipients suchas fillers, extenders, binders, humectants, disintegrating agents andsurfactants; or diluents, which are commonly used for preparingformulations depending on usage forms. The dosage form of thepharmaceutical composition of the present invention can include, but notlimited to, e.g. tablets, powders, granules, and pills, or solutions,suspensions and emulsions, so far as it effectively contains a peptiderepresented by the formula (I). Furthermore, the pharmaceuticalcomposition of the present invention may be dry forms capable ofchanging into a liquid form by addition of a proper carrier before use.The formulation can be carried out in a usual manner.

Dose of the pharmaceutical composition of the present invention isappropriately selected depending on administration methods, dosageforms, or conditions of a patient to be administered, or the like.Generally, it is appropriate to prepare a formulation comprising apeptide of the invention at a rate of about 1-100% by weight, and toadminister the peptide contained in the formulation in a dose of about0.2-300 mg per day per adult person. Furthermore, the administration isnot necessarily given once a day, but can be divided over several timesa day.

The pharmaceutical composition of the invention can be administered viaan administration route which depends on dosage forms. For example, inthe case of an injection form, administration can be intravenous,intramuscular, subcutaneous, intracutaneous, or intraperitoneal, and inthe case of a solid form, administration can be done perorally, etc.

The pharmaceutical composition of the invention has food and waterintake inhibitory effects, as well as weight-reducing andobesity-inhibiting effects. Hence, the administration of thepharmaceutical composition of the invention results in food and waterintake inhibitory effects, and accompanying weight-reducing andobesity-inhibiting effects, indicating that the composition isparticularly useful for diseases such as obesity.

The specification incorporates the descriptions in the specification ofJapanese Patent Application No. 10-172325 on which the priority of thepresent application is based.

BEST MODES FOR CARRYING OUT THE INVENTION Example 1

Weight-reducing Effect

PeptideAla-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Val-Ala-Ala-Ala-Asn-Try-Lys-Lys-Pro-Lys-Leu(SEQ ID NO:3) was synthesized according to Lin Y. Z. et al., J. Biol.Chem. 270, 14255-14258 (1995); Merrifield, R. B. J. Am. Chem. Soc. 85,2149-2154 (1963); and Lin Y. Z. et al., Biochemistry 27, 5640-5645(1988). In the amino acid sequence of this peptide,Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Val (SEQ IDNO: 1) corresponds to the membrane-transfer amino acid sequence,Ala-Ala-Ala to the linker sequence, and Asn-Try-Lys-Lys-Pro-Lys-Leu (SEQID NO:2) to the nuclear localization amino acid sequence, respectively.

This peptide was dissolved in physiological saline to prepare a 2.6pmol/μl peptide-containing injection solution. Five μl of the injectionsolution (containing 13 pmol peptide) was administered into the cerebralventricles of 10-week-old male Wistar rats, which were weighed on thenext day to calculate amounts of change in weight prior to andsubsequent to injection.

On the other hand, 5 μl physiological saline was administered to rats asa control in the same manner as described above, and 2.6 pmol/μlFGF-containing injection solution was administered to rats as acomparative example in the same manner as described above. Each rat wasweighed on the next day to calculate amounts of change in weight priorto and subsequent to injection.

The results are shown in Table 1.

TABLE 1 Administration Amount of substance dose change in weightPhysiological 5 μl +2.0 g saline FGF 13 pmol/5 μl −10.1 g Peptide 13pmol/5 μl −5.3 g

As shown in Table 1, the weights of the rats to which the peptide wasadministered, were significantly reduced when compared to those of therats to which the physiological saline as a control was administered.Furthermore, the weights of the rats to which the peptide wasadministered, were reduced almost equally when compared to those of therats to which FGF was administered.

From these results, it was demonstrated that the peptide of theinvention has a weight-reducing effect.

Example 2

Food and Water Intake Inhibitory Effects

In the same manner as in example 1, the peptideAla-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Val-Ala-Ala-Ala-Asn-Try-Lys-Lys-Pro-Lys-Leu(SEQ ID NO:3) was synthesized. This peptide was dissolved inphysiological saline to prepare a 2.6 pmol/μl peptide-containinginjection solution. Five μl of the injection solution (containing 13pmol peptide) was administered in the cerebral ventricles of 10-week oldmale Wistar rats, and the food and water intake level were measured 12hours after administration to compare with those in the same period ofthe day prior to administration.

On the other hand, 5 μl physiological saline was administered as acontrol in the same manner as described above, and 2.6 pmol/μlFGF-containing injection solution was administered as a comparativeexample in the same manner as described above. Food and water intakelevels were measured respectively 12 hours after administration, andwere compared to those in the same period of the day prior toadministration to calculate amounts of change in food and water intake.

The results are shown in Table 2.

TABLE 2 Amount of Amount of Administration change in food change insubstance dose intake water intake Physiological 5 μl −1.1 g −0.3 mlsaline FGF 13 pmol/5 μl −6.9 g −12.2 ml Peptide 13 pmol/5 μl −4.8 g −8.4ml

As shown in Table 2, food and water intake levels were significantlyreduced in the rats to which the peptide was administered when comparedto those of the control rats to which the physiological saline wasadministered. Furthermore, the food and water intake levels of the ratsto which the peptide was administered were reduced almost equally whencompared to those of the rats to which FGF was administered.

From these results, it was demonstrated that the peptide of theinvention has food and water intake inhibitory effects.

All references cited herein are incorporated by reference in theirentirety in the present specification.

Industrial Applicability

This invention provides a pharmaceutical composition having food andwater intake inhibitory effects, and accompanying weight-reducing andobesity-inhibiting effects. The administration of the pharmaceuticalcomposition of the invention results in food and water intake inhibitoryeffects, as well as weight-reducing and obesity- inhibiting effects,indicating that the composition is particularly useful for diseases suchas obesity.

Furthermore, the pharmaceutical composition of the invention comprises,as an active ingredient, a peptide with superior cell membrane transferand cell nucleus membrane transfer, and it can be used as a carrier fortransporting other drugs to the cytoplasm or cell nuclei.

3 1 16 PRT Artificial Sequence Derivative of partial peptide offibroblast growth factors 1 Ala Ala Val Ala Leu Leu Pro Ala Val Leu LeuAla Leu Leu Ala Val 1 5 10 15 2 7 PRT Artificial Sequence Derivative ofpartial peptide of fibroblast growth factors 2 Asn Tyr Lys Lys Pro LysLeu 1 5 3 26 PRT Artificial Sequence Derivative of partial peptide offibroblast growth factors 3 Ala Ala Val Ala Leu Leu Pro Ala Val Leu LeuAla Leu Leu Ala Val 1 5 10 15 Ala Ala Ala Asn Tyr Lys Lys Pro Lys Leu 2025

What is claimed is:
 1. A pharmaceutical composition comprising a peptiderepresented by the following formula (I): MTS-X-NLS  (I) wherein saidMTS represents a membrane-transfer amino acid sequence and isrepresented by an amino acid sequence of SEQ ID NO:1; X represents adirect bond or a linker sequence; and said NLS represents a nuclearlocalization amino acid sequence and is represented by an amino acidsequence of SEQ ID NO:2; or a pharmaceutically acceptable salt thereofas an active ingredient.
 2. A method of reducing food and/or waterintake comprising administering a pharmaceutical composition comprisingthe composition of claim 1, whereby the composition reduces food and/orwater intake.